JPS59184132A - Production of pertussis vaccine - Google Patents

Abstract

PURPOSE:To obtain the titled vaccine on an industrial scale, by cultivating Bordetella pertussis with aeration and agitation under specific conditions, increasing an infection protective antigen HA fraction together with the multiplication of the Bordetella pertussis, and detoxicating the above-mentioned HA fraction in the presence of an amino acid. CONSTITUTION:Bordetella pertussis is inoculated into a liquid culture medium containing cyclodextrin or a derivative thereof. In the process, the cultivation temperature is kept at 20-37 deg.C, and the amount of dissolved oxygen in the culture medium is kept at 0.7-6ppm. Furthermore, the culture medium is preferably defoamed under conditions of 6<pH<9 for the cultivation with aeration and agitation. The aimed infection protective antigen HA fraction is then collected in the bacterial growth stage in the ogarithmic growth phase or stationary phase and then detoxicated in the presence of an amino acid, e.g. glycine or methionine. The resultant detoxicated HA fraction can be used to produce purified pertussis vaccine, precipitated purified pertussis vaccine, precipitated purified pertussis and diphtheris and tetanus mixed vaccine, etc. economically in large amounts.

Description

【発明の詳細な説明】 本発明は、百日ぜき菌の感染防御抗原ＨＡ画分（Ｆ　−
ＨＡ　：　Ｆｉｌａｍｅｎｔｏｕｓ　Ｈｅｍａｇｇｌｕ
ｔｉｎｉｎおよびＬ　Ｐ　Ｅ　−ＨＡ　：　Ｌｅｕｃｏ
ｃｙｔｏｓｉｓ−ｐｒｏｍｏｔｉｎｇＦａｃｔｏｒ　Ｈ
ｅｍａｇｇｌｕｔｉｎｉｎを含んだ両分）を採取し、該
ＨＡ画分をアミノ酸の存在下に無毒化することによ・９
百日ぜきワクチンを製造する方法、さらに詳しくは、百
日ぜき菌をシクロデキストリン寸たはその誘導体を添加
した液状培地にて通気攪拌培養するに際し、培養温度お
よび溶存酸素量を特定範囲に制御しかつ消泡条件下で行
なうことにより百日ぜき菌の感染防御抗原ＨＡ画分を採
取し、これをアミノ酸の存在下に無毒化して百日ぜきワ
クチンを工業的規模にて製造する方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of the infection-protective antigen HA fraction of Bordetella pertussis (F-
HA: Filamentous Hemaglu
tinin and LPE-HA: Leuco
cytosis-promotingFactor H
By collecting both fractions containing emaggglutinin and detoxifying the HA fraction in the presence of amino acids, 9
A method for producing a pertussis vaccine, more specifically, when culturing Bordetella pertussis with aeration in a liquid medium supplemented with cyclodextrin or its derivatives, the culture temperature and amount of dissolved oxygen are controlled within a specific range, and antifoaming conditions are used. This invention relates to a method for producing a pertussis vaccine on an industrial scale by collecting a protective antigen HA fraction of Bordetella pertussis and detoxifying it in the presence of amino acids.

産業上の利用分野 百日ぜきは我が国では届出伝染病に指定されてお９、乳
児〜幼児に多発する公衆衛生上重要な感染症である。と
くに乳児では重症経過をたどることが多く、時には死亡
例もみられる。この疾病は古くからワクチンによる予防
が効果的であることが知られており、原因菌である百日
ぜき菌■−相菌の全菌体の不活性ワクチンが広く用いら
れていた。Industrial applications Pertussis is designated as a notifiable infectious disease in Japan9 and is an important infectious disease from a public health perspective that frequently affects infants and young children. Infants in particular often develop a severe course, and sometimes even die. It has been known for a long time that this disease can be effectively prevented by vaccination, and an inactivated vaccine made from the whole body of the causative bacteria, Bordetella pertussis -, has been widely used.

しかし、このような菌体不活化ワクチンは副作用が強く
、そのため一時期にはワクチンの接種が中止されていた
。その一方、百日ぜきによる乳幼児の疾病は大きな問題
となってお９、副作用のないワクチンの製造が熱望され
ていた。However, such inactivated bacterial vaccines have strong side effects, and as a result, vaccination was discontinued for a period of time. On the other hand, illness in infants and children caused by pertussis has become a serious problem9, and there has been a strong desire to produce a vaccine without side effects.

従来技術 先に、佐藤らは感染防御抗原に関する基礎的研究をもと
にして画期的なコンポーネントワクチンである沈降百日
ぜき精製ワクチンの製造に成功した（特公昭５７−５２
０３号を参照）。このワクチンはＦ−ＨＡおよびＬＰＦ
−ＨＡを含んだＩ−Ｉ　Ａ画分を主な感染防御抗原とし
、副作用をほとんど示すことなく優れた予防効果を有す
るものであってすでに実用化されている。Conventional technology Previously, Sato et al. succeeded in producing a purified precipitated pertussis vaccine, an epoch-making component vaccine, based on basic research on infectious protective antigens (Special Publication No. 57-52).
(See issue 03). This vaccine is F-HA and LPF
-The I-IA fraction containing HA is used as the main infection-protective antigen, and it has an excellent preventive effect with almost no side effects and has already been put into practical use.

この実用化されているワクチンの製造には、百日ぜき■
相菌を適当な培地に接種し、３５°Ｃ前後で５日間静置
培養し、培養液を遠心し、その上清に硫酸アンモニウム
を約５０チ飽和になるように加えるかアルコール添加し
、生じた沈殿を１０．ＯＱ　Ｑ　ｒｐｍ、３０分間遠心
して分離し、この沈殿を塩化ナトリウム添加緩衝液にて
抽出し、その抽出画分を常法によりショ糖密度勾配遠心
にかけて百日ぜきＩ（Ａ画分を回収し、ホルマリンで無
毒化処理してワクチンとしており、所望によりこれにジ
フテリアトキソイド、破傷風トキソイドを加え、さらに
必要によりアルミニウムアジュバント処理し、ゼラチン
、グルコースなどの安定剤を添加して沈降精製百日ぜき
・ジフテリア・破傷風混合ワクチンとしている。The production of this commercially available vaccine involves pertussis■
The compatible bacteria was inoculated into an appropriate medium, cultured for 5 days at around 35°C, the culture solution was centrifuged, and ammonium sulfate was added to the supernatant to saturation of about 50%, or alcohol was added to the resulting supernatant. Precipitate 10. The precipitate was separated by centrifugation at OQ Q rpm for 30 minutes, and the precipitate was extracted with a buffer solution containing sodium chloride. The vaccine is detoxified and diphtheria toxoid and tetanus toxoid are added to it, if necessary, and aluminum adjuvant treatment is added if necessary. Stabilizers such as gelatin and glucose are added to produce a pertussis/diphtheria/tetanus combined vaccine that is purified by precipitation. There is.

しかしながら、この方法ではとくに培養に難点があり、
大規模な培養が不可能でワクチンの量産が困難である。However, this method has some difficulties, especially in culturing.
Large-scale cultivation is not possible, making mass production of vaccines difficult.

すなわち、この公知の沈降百日ぜき精製ワクチンの製造
法では、ル−瓶などの小容器に液状培地を１００〜３０
０ｍｌ！程度入れて横臥位置で３５℃前後にて５日間静
置培養するもので、きわめて小規模でかつ長期間を要す
る。一般に微生物の大量培養には液状培地による攪拌培
養方式が採用されることが多い。百日ぜき菌は液状培地
による振盪培養を行なうと菌自身の増殖はある程度まで
は達成されるが、たとえばＨＡ画分の産生けきわめて低
いといわれている（　Ａｒａｉ、　Ｈ，＆Ｍｕｎｏｚ、
　Ｊ、　Ｊ、、　Ｉｎｆｅｃｔ、　Ｉｍｍｕｎ、　２５
．　７６４−７６Ｌ、’　１９７９を参照）。このこと
は精製ワクチンの構成成分の少なくとも一方は量産し難
いことを示唆するものである。しだがって、この佐藤ら
の百日ぜきワクチンは画期的なワクチンであるがその製
造には小規模で長時間を要する静置培養に頼らざるを得
す、その製法の改良が熱望されている。That is, in this known method for producing a purified precipitated pertussis vaccine, 100 to 30% of a liquid medium is placed in a small container such as a Lou bottle.
0ml! The culture is statically cultured in a recumbent position for 5 days at around 35°C, which requires a very small scale and a long period of time. Generally, a stirring culture method using a liquid medium is often adopted for mass culture of microorganisms. When B. pertussis is cultured with shaking in a liquid medium, the bacterium itself can grow to a certain extent, but it is said that the production of HA fraction, for example, is extremely low (Arai, H, & Munoz,
J, J,, Infect, Immun, 25
．． 764-76L, '1979). This suggests that at least one of the components of a purified vaccine is difficult to mass produce. Therefore, although Sato et al.'s pertussis vaccine is a groundbreaking vaccine, its production must rely on static culture, which is small-scale and takes a long time, and improvements to the production method are eagerly awaited. .

最近、鈴木らは百日ぜき■相菌の増殖を促進しかつＬＰ
Ｆ−ＨＡの産生を促進しつる添加物の検索ヲ試み、シク
ロデキストリンおよびその誘導体、とくにメチル化β−
シクロデキストリン（２，６−ジ（０−メチル）−β−
シクロデキストリン、以下メチル化β−ＣＤと略称する
）の添加が百日ぜき■相菌のステイナーショルテ液体培
地（Ｓｔａｉｎｅｒ。Recently, Suzuki et al.
An attempt was made to search for additives that promote the production of F-HA, including cyclodextrin and its derivatives, especially methylated β-
Cyclodextrin (2,6-di(0-methyl)-β-
Cyclodextrin (hereinafter abbreviated as methylated β-CD) is added to the pertussis ■ Stainer Scholte liquid medium (Stainer).

Ｄ、　Ｗｌ＆８ｃｈｏｌｔｅ、　Ｍ、　Ｊ、　；　Ｊ、
　Ｇｅｎ、　Ｍｉｃｒｏｂ、ｔｏｌ、　６３　。D, Wl&8cholte, M, J;
Gen, Microb, tol, 63.

２１１−２２０．１９７１を参照）を用いた攪拌培養に
おける菌増殖およびＬＰＦ−ＨＡ産生を促進すること、
さらに培養液中でのＬＰＦ−ＨＡの安定性にも寄与する
ことを報告している（鈴木ら、第２９回毒素シンポジウ
ム予稿集、１〜５．１９８２を参照）。211-220.1971)) to promote bacterial growth and LPF-HA production in agitation culture;
Furthermore, it has been reported that it also contributes to the stability of LPF-HA in the culture solution (see Suzuki et al., Proceedings of the 29th Toxin Symposium, 1-5, 1982).

しかしながら、かかる方法を１０１あるいはそれ以上の
スケールの発酵槽を用いる工業的規棟の百日ぜきワクチ
ンの製造に適用した場合には、従来の攪拌培養にもとづ
く知見からは全く類推できない結果が得られた−０すな
わち、攪拌条件を一定とする振盪培養や攪拌培養系では
菌数の増加は見られる場合もあるが、ＬＰＦ　　ＨＡの
産生量は充分でないことを知ったのである。However, when this method was applied to the production of pertussis vaccine on an industrial scale using fermenters of 101 or more scale, results were obtained that could not be inferred from the knowledge based on conventional agitation culture. In other words, we learned that although an increase in the number of bacteria may be observed in shaking culture or stirring culture systems where stirring conditions are constant, the amount of LPF HA produced is not sufficient.

まだ、ＷＨＯの１９７７年刊行の資料（Ｍａｎｕａｌｆ
ｏｒ　ｔｈｅ　ｐｒｏｄｕｃｔｉｏｎ　ａｎａ　ｃｏｎ
ｔｒｏｌ　ｏｆ　ｖａｃｃｉｎｅｓＰθｒｔｕｓＳｉｅ
　ｖａｃｃｉｎｅ、　ＷＨＯを参照）によれば、百日ぜ
きワクチンの製法に関して発酵槽を用いた百日ぜき菌の
大量培養について記載されており、空気を上方からの表
面通気によりあるいはグリッドを通して培地中に入れ、
特殊な羽根で攪拌して培養液内に巻込む方式で、一定の
通気攪拌によって百日ぜき菌菌体を得ることができると
している。Still, the WHO's 1977 publication (Manual
or the production ana con
Trol of vaccinesPθrtusSie
Vaccine (see WHO) describes the mass cultivation of Bordetella pertussis using fermenters for the preparation of pertussis vaccine, in which air is introduced into the medium by surface aeration from above or through a grid;
The company says it is possible to obtain pertussis bacteria by stirring with a special blade and incorporating it into the culture solution, with constant aeration and agitation.

しかしながら、本発明者らは、ステイナー・ショルテ培
地あるいは後述のその改良培地１０１の培養規模におい
てＷＨＯの記述に準じ、槽底からの通気量を０，２ＶＶ
Ｍ（空気流量（Ｉｌｌ’）／培養容量（１）／時間＠）
、羽根の回転数を５００あるいは６００ｒｐｍの一定と
し、いわゆる槽底からの一定通気攪拌培養系について検
討を加えたところ、菌数の増加は期待できるが、百日ぜ
き菌ＨＡ画分の産生は不充分であシ、到底、精製百日ぜ
きワクチンの工業的生産には適さないことを知った。However, the present inventors have determined that the aeration rate from the bottom of the tank is 0.2 VV in accordance with the WHO description on the culture scale of Steiner-Scholte medium or its improved medium 101 described below.
M (air flow rate (Ill') / culture volume (1) / time @)
When we investigated a so-called constant aeration agitation culture system from the bottom of the tank in which the rotational speed of the blades was kept constant at 500 or 600 rpm, an increase in the number of bacteria could be expected, but the production of the Bordetella pertussis HA fraction was insufficient. I learned that it was completely unsuitable for industrial production of purified pertussis vaccine.

そこで、本発明者らは、大規模な培養装置、とくに通常
の発酵槽を用いた通気攪拌培養においても菌の増殖とと
もに所望のＨＡ画分の大量生産に適した培養条件を見い
出すべく種々研究を重ねた結果、ある範囲の培養温度に
おいて溶存酸素量（以下、Ｄｏと略記することがある）
を特定の範囲に制御しかつ消泡処理をしながら、さらに
望ましくは、ｐＨの制御条件下に培養することにより、
大規模な培養、とくに通常の発酵槽を用いた通気攪拌培
養においても、百日ぜき菌の著しい増殖とともに、百日
ぜき菌ＨＡ画分を著しく増大しうろことを見い出し、さ
らにこのＨＡ画分にアミノ酸の存在下で通常の不活化剤
、例えばホルマリンやグルタルアルデヒドを加えること
により無毒化が達成され、３７℃長期間加温経過しても
毒性復帰現象（リバース）がおこらないことを見出した
。この無毒化処理におけるアミノ酸の添加系ではアミノ
酸非添加系に比して凝集塊沈殿を生成することはなく、
音波処理等の工程が不要でそのまま無毒化Ｉ（Ａ画分を
メンブレンフィルターを用いて無菌濾過できることを見
出し、本発明を完成するに至った。Therefore, the present inventors conducted various studies in order to find culture conditions suitable for bacterial growth and mass production of the desired HA fraction even in aerated agitation culture using a large-scale culture apparatus, especially a normal fermenter. As a result of stacking, the amount of dissolved oxygen (hereinafter sometimes abbreviated as Do) at a certain range of culture temperature
By controlling the pH within a specific range and performing antifoaming treatment, and more preferably culturing under pH controlled conditions,
Even in large-scale culture, especially in aerated agitation culture using a normal fermenter, we found that scales significantly increased the HA fraction of Bordetella pertussis and the presence of amino acids in this HA fraction. It was found that detoxification was achieved by adding a conventional inactivating agent such as formalin or glutaraldehyde, and that no reversal of toxicity occurred even after long-term heating at 37°C. In this detoxification treatment, the amino acid addition system does not produce aggregate precipitates compared to the amino acid non-addition system.
We have discovered that the detoxified I (A fraction) can be sterile-filtered directly using a membrane filter without the need for steps such as sonication, and have completed the present invention.

発明の構成および効果 本発明によれば、百日ぜき菌をシクロデキストリンまた
はその誘導体を添加した液状培地に接種し、消泡処理し
ながら、培養温度２０〜３７℃において溶存酸素量を０
．７〜６．０ｐｐｍの範囲に保ち、さらに望ましくはｐ
Ｈをたとえば６０〜９．０にて通気攪拌培養し、対数増
殖期ないし定常期の菌発育段階で感染防御抗原ＨＡ画分
を採取し、該ＨＡ画分をアミノ酸の存在下でホルマリン
やグルタルアルデヒドなどで無毒化し、これを用いて所
望の精製百日ぜきワクチン、沈降精製百日ぜきワクチン
、沈降精製百日ぜき・ジフテリア・破傷風混合ワクチン
等が大量かつ経済的に製造される。Structure and Effects of the Invention According to the present invention, Bordetella pertussis is inoculated into a liquid medium supplemented with cyclodextrin or its derivative, and the amount of dissolved oxygen is reduced to 0 at a culture temperature of 20 to 37° C. while being treated with defoaming.
．． Keep it within the range of 7 to 6.0 ppm, more preferably p
For example, culture H with aeration at a temperature of 60 to 9.0, collect the infectious protective antigen HA fraction at the logarithmic growth phase or stationary growth stage, and incubate the HA fraction with formalin or glutaraldehyde in the presence of amino acids. Using the same, desired purified pertussis vaccines, precipitated purified pertussis vaccines, precipitated purified pertussis/diphtheria/tetanus combined vaccines, etc. can be produced economically in large quantities.

本発明で用いられる百日ぜき菌株としては、通常ワクチ
ン株として知られているものであればいずれでもよく、
一般にその■相菌のボルデ・ジャング培地継代菌あるい
は振盪培養菌が用いられ、これを種菌として液状培地に
接種する。なお、百日ぜき菌■相菌も適用することがで
きる。接種量はとくに限定されないが、通常、最終濃度
が０．２〜ｌ　Ｑ　ＩＯＵ／ｆｎｌ（ＩＯＵ：Ｉｎｔｅ
ｒｎａｔｉｏｎａｌ　ｏｐａｃｉｔｙｕｎｉｔ、生物学
的製剤基準、２３８．１９７９．厚生省を参照）、好ま
しくは約１．　□　ＩＯＵ／ｍＡとなる程度である。The pertussis strain used in the present invention may be any strain commonly known as a vaccine strain.
Generally, a subcultured Bordet-Jung medium or a shake-cultured microorganism of the phase fungus is used, and this is inoculated into a liquid medium as a seed microorganism. In addition, Bordetella pertussis can also be applied. The amount of inoculation is not particularly limited, but the final concentration is usually 0.2 to 1 Q IOU/fnl (IOU: Inte
National Opacity Unit, Biological Products Standard, 238.1979. (see Ministry of Health and Welfare), preferably about 1. □ IOU/mA.

液状培地としては公知のいずれの培地も用いられるが、
好ましくはステイナー・ショルテ培地、とくに好ましく
は、該ステイナー・ショルテ培地を基本とし、これにカ
ザミノ酸を０１〜２０１１／１添加シ、アスコルビン酸
を０．０１〜Ｉｆ／ｌ、グルタチオンを０．１〜５９／
１３の範囲に調整したステイナー・ショルテ改良培地（
以下、単に改良培地という）が用いられる。Any known medium can be used as the liquid medium, but
Preferably, the Steiner-Scholte medium is used as a base, and particularly preferably, the Steiner-Scholte medium is used as a base, with addition of casamino acids from 01 to 2011/1, ascorbic acid from 0.01 to If/l, and glutathione from 0.1 to 1/1. 59/
Steiner-Scholte modified medium adjusted to a range of 13 (
(hereinafter simply referred to as an improved medium) is used.

培地に添加されるシクロデキストリン（以、下、ＣＤと
略称する）またはその誘導体としては、α−ＣＤ、β−
ＣＤ、γ−ＣＤなどの異性体、メチル化α−ＣＤ、メチ
ル化β−ＣＤ（前掲）、メチル化γ−ＣＤなどのエーテ
ル化誘導体のほか、アミン化誘導体かエステル化誘導体
などが挙げら江それらは単独で捷だけ２種以上を併用し
て用いられる・これらのうち、メチル化β−ＣＤがもつ
とも良好な添加効果を示す。その添加量はとくに限定さ
れないが、通常、０００１〜５１１／１１．好ましくは
約０．５〜２．５ｆ／１１である。Cyclodextrin (hereinafter abbreviated as CD) or its derivatives added to the medium include α-CD, β-
Examples include isomers such as CD and γ-CD, etherified derivatives such as methylated α-CD, methylated β-CD (listed above), and methylated γ-CD, as well as aminated derivatives and esterified derivatives. They can be used alone or in combination of two or more types.Among these, methylated β-CD shows the best addition effect. The amount added is not particularly limited, but is usually 0001 to 511/11. Preferably it is about 0.5 to 2.5 f/11.

本発明者らは百日ぜき菌の大規模培養における菌増殖、
Ｆ−ＨＡおよびＬ　Ｐ　Ｆ−ＨＡ産生量の増大には培養
温度ならびにＤｏの制御が大きな要因となることを初め
て認め、かつ消泡操作の有無さらには培地のｐＨの制御
も大きく影響することを明らかにした。これらの成績に
ついて以下説明する。The present inventors demonstrated bacterial growth in large-scale culture of Bordetella pertussis,
It was recognized for the first time that the control of culture temperature and Do is a major factor in increasing the production of F-HA and LPF-HA, and that the presence or absence of defoaming operation and control of the pH of the culture medium also have a large effect. revealed. These results will be explained below.

培養温度については、百日ぜき菌Ｉ相菌東浜株をボルデ
・ジャング培地で継代したものを種菌とし、メチル化β
−ＯＤ　］、、　Ｏｆ／ｌを添加した改良培地１０ｍｇ
にＱ、　２　ＩＯＵ／ｍ６になるように接種し、温度勾
配培養装置ＴＮ１１２Ｄ（東洋科学産業製）を用い、培
養温度を１７℃から４２°Ｃの範囲で振盪速度６０回／
分にて４８時間振盪培養して至適−範囲を調べた。増殖
した菌数は光電比色計コゝ−ルマンジュニア６Ｄ型（コ
ールマン社製）ヲ用イ、０Ｄ６５０における測定値から
換算して求めた。Regarding the culture temperature, the inoculum was the Bordetella pertussis I phase bacteria Higashihama strain subcultured in Bordet-Jung medium, and the methylated β
-OD ],, 10 mg of improved medium supplemented with Of/l
Q, 2 IOU/m6 was inoculated, and using a temperature gradient culture device TN112D (manufactured by Toyo Kagaku Sangyo), the culture temperature was set in the range of 17°C to 42°C, and the shaking rate was 60 times/.
The optimum range was determined by culturing with shaking for 48 hours. The number of proliferated bacteria was determined by converting the measured value using a photoelectric colorimeter Coleman Junior 6D model (manufactured by Coleman Co., Ltd.) at 0D650.

なお、この実験は実験室的小規模にて振盪培養で行なっ
たが、培養温度に関しては大規模な通気攪拌培養でも同
傾向を示す。Although this experiment was carried out using shaking culture on a small scale in the laboratory, the same tendency can be observed in large-scale aerated agitation culture with regard to culture temperature.

その結果を第１図に示しだが、菌の増殖は２０〜３７℃
の範囲が望ましく、より好捷しくけ２３〜３７℃であっ
た。The results are shown in Figure 1, and the growth of bacteria was between 20 and 37 degrees Celsius.
A range of 23 to 37°C is preferable, and a more favorable range is 23 to 37°C.

培地の溶存酸素量（ＤＯ）は０．７〜６．０　ｐｐｍ、
好ましくは１０〜５．５　ｐｐｍの範囲に保持される。The amount of dissolved oxygen (DO) in the medium is 0.7 to 6.0 ppm,
Preferably it is maintained within the range of 10 to 5.5 ppm.

この範囲内に制御することによシ、百日ぜき菌の増殖が
増大するとともに、所望のＬＰＦ−ＨＡおよびＦ−ＨＡ
の産生も著しく増大する。By controlling within this range, the growth of Bordetella pertussis is increased and the desired LPF-HA and F-HA are
The production of is also significantly increased.

なお、ＤＯ制御には通気量と攪拌速度の制御を組合せて
行なうのがもつともよく、通気量と攪拌速度はとくに限
定されないが、通常の通気攪拌槽を用いた場合には、空
気の通気量は３ＶＶＭ以下、通常０１〜２ＶＶＭ、好ま
しくは０．１〜１．５ＶＶＭの範囲であり、攪拌速度は
６００　ｒｐｍ以下、通常５０〜３５０　ｒｐｍ、好ま
しくは１００〜２５０ｒｐｍの範囲である。ただし、純
酸素を併用する場合は通気量あるいは攪拌速度は減する
ことができる。Note that DO control may be carried out in combination with control of the aeration amount and stirring speed, and the aeration amount and stirring speed are not particularly limited. However, when using a normal aeration stirring tank, the amount of air aeration is The stirring speed is 3 VVM or less, usually 01 to 2 VVM, preferably 0.1 to 1.5 VVM, and the stirring speed is 600 rpm or less, usually 50 to 350 rpm, preferably 100 to 250 rpm. However, if pure oxygen is used in combination, the aeration amount or stirring speed can be reduced.

また、消泡操作の有無によっても培養液中の菌数増加お
よびＦ−ＨＡ、ＬＩ’Ｆ−ＨＡ量の収率が大きく影響さ
れ、後述の実施例１と同様の実験条件で培養した場合、
消泡を行わないときには泡に付着した菌体がそのまま槽
壁に累積されたり、排気ノズルから流出されたりして培
養液中の菌数、ＦＨＡおよびＬＰＦ−ＨＡ量ともに数〜
８０％程度の派別が認められた。なお、消泡は機械的消
泡と化学的消泡剤のいずれも適用され、例えば回転ティ
スフ式、スプレーノズル方式などの公知の消泡用装置を
用いるか、あるいは通常の脂肪酸エステル系、シリコン
系、アルコール系などの化学的消泡剤を用いることがで
きる。なお、培養液からのＨＡ画分の採取、精製等の点
からは機械的消泡手段を用いるのがよシ好ましい。In addition, the increase in the number of bacteria in the culture solution and the yield of F-HA and LI'F-HA are greatly influenced by the presence or absence of defoaming operation, and when cultured under the same experimental conditions as in Example 1 described below,
If defoaming is not performed, the bacterial cells attached to the foam may accumulate on the tank wall or be discharged from the exhaust nozzle, resulting in a decrease in the number of bacteria in the culture solution and the amount of FHA and LPF-HA.
Approximately 80% sectarianism was recognized. For defoaming, both mechanical defoaming and chemical defoaming agents can be applied; for example, known defoaming devices such as a rotary tisf type or a spray nozzle system may be used, or ordinary fatty acid ester-based or silicon-based defoaming agents may be used. Chemical antifoaming agents such as , alcohol-based, etc. can be used. Note that from the viewpoint of collection and purification of the HA fraction from the culture solution, it is more preferable to use mechanical antifoaming means.

培地ｐＨの至適範囲を知るため、ｐＨを種々に変えて菌
の増殖を調べた。ＤＯを２．５　ｐｐｍと一定にした以
外は後述の実施例１と同様の実験条件で培養した。ｐＨ
６，０〜９．０の範囲ではいずれも菌増殖は達せられ、
ｐＨ５，５〜８．５、とくにｐＨ６，８〜７５の範囲で
は菌増殖速度が若干増大することが認められた。In order to find out the optimal range of medium pH, bacterial growth was examined by varying the pH. Culture was carried out under the same experimental conditions as in Example 1 described below, except that the DO was kept constant at 2.5 ppm. pH
Bacterial growth was achieved in the range of 6.0 to 9.0,
It was observed that the bacterial growth rate increased slightly in the range of pH 5.5 to 8.5, particularly pH 6.8 to 75.

本発明による培養温度、溶存酸素量さらには消泡、ｐｎ
などの制御は自動制御および手動制御のいずれも採用さ
れる。Culture temperature, dissolved oxygen amount, defoaming, pn
Both automatic control and manual control are adopted.

また、目的とするＨＡ画分を高収率で得るには菌の培養
状態のチェックが重要であり、対数増殖期から転換期を
経て定常期に至るまでの菌発育段階において採取するの
がもつとも望ましく、それは接種菌によって変るが、通
常、７〜４０時間に相当し、例えば、１．０　ＩＯＵ／
ｍｄの接種菌量の場合には通常２４〜３５時間である。In addition, in order to obtain the desired HA fraction with a high yield, it is important to check the culture state of the bacteria, and it is important to collect the bacteria during the growth stage from the logarithmic growth phase through the conversion phase to the stationary phase. Desirably, it varies depending on the inoculum, but usually corresponds to 7 to 40 hours, e.g. 1.0 IOU/
In the case of an inoculum amount of md, it is usually 24 to 35 hours.

上記のようにして産生されるＨＡ含有培養液から沈降精
製百日ぜきワクチンが調製される。A sedimentation-purified pertussis vaccine is prepared from the HA-containing culture solution produced as described above.

すなわち、得られた培養液には直接または連続遠心処理
したのち、硫酸アンモニウムを約１／３飽和になるよう
に加え、生じた沈殿を遠心分離まだは濾過して収集し、
これを１モル塩化ナトリウム添加リン酸緩衝液、ｐＨ７
，２に溶解する。この溶液に硫酸アンモニウムを約１／
２飽和になるまで加え、生じた沈殿を遠心分離または濾
過などにより収集し、これを透析チューブに入れて１モ
ル塩化ナトリウム添加リン酸緩衝液、　ｐＨ７，２に対
して透析し溶解する。これを超遠心分離にかけ、得られ
る上清をさらにショ糖密度勾配遠心にかけて、その上清
（百日ぜき菌ＨＡ画分）を得る。これらの一連の精製工
程は４°Ｃ以下で行なうのが望ましい。得られた上清に
は、大量のＬＰＦ−ＨＡとＦ−ＨＡが含まれている。こ
れを電気泳動法により調べたところ、従来の静置培養法
由来のＦ−ＨＡやＬＰＦ−ＨＡと同程度の分子量および
電荷をもち、さらに同等の形態（電子顕微鏡所見）、抗
原性（ゲル内沈降反応所見）およびマウスＬＰＦ毒素活
性を有している。That is, after directly or continuously centrifuging the obtained culture solution, ammonium sulfate is added to about 1/3 saturation, and the resulting precipitate is collected by centrifugation or filtration.
This was added to a phosphate buffer solution containing 1M sodium chloride, pH 7.
,2. Add about 1/2 ammonium sulfate to this solution.
2 to saturation, and the resulting precipitate is collected by centrifugation or filtration, placed in a dialysis tube, and dialyzed against 1M sodium chloride-added phosphate buffer, pH 7.2, and dissolved. This is subjected to ultracentrifugation, and the resulting supernatant is further subjected to sucrose density gradient centrifugation to obtain the supernatant (B. pertussis HA fraction). These series of purification steps are desirably carried out at 4°C or below. The obtained supernatant contains a large amount of LPF-HA and F-HA. When examined by electrophoresis, it was found to have the same molecular weight and charge as F-HA and LPF-HA derived from conventional static culture, as well as the same morphology (electron microscopy findings) and antigenicity (in the gel). (precipitation reaction findings) and mouse LPF toxin activity.

このＨＡ画分を適宜希釈し、これにホルマリンをＯ，’
１〜１．２Ｖ／ｖ％、好ましくは０４〜０．８ｖ／ｖチ
の濃度に添加し、２０〜４３”Ｃ１好ましくは３７〜４
０℃で５〜６０日間処理する。このホルマリン無毒化処
理によ５ＨＡ画分中のＬＰＦ活性、Ｈ８Ｆ活性（Ｈｉｓ
ｔａｍｉｎｅ　−ｓｅｎｓｉｔｉｚｉｎｇ　ｆａｃｔｏ
ｒ　）等が減車される。本発明者らは、この無毒化処理
においてアミノ酸を添加すれば、無毒化に要する時間が
著しく短縮され、凝集塊沈殿を生成することなく、毒性
復帰現象（リバース）が起こらないことを見出した。な
お、この際アミノ酸とともに安定剤としてツイーン８０
、ゼラチンを適宜添加することができる。グルタルアル
デヒドの場合は、０．０５〜０３ｖ／ｖ％の濃度に添加
し、室温で１〜７日間処理するのが望ましい。This HA fraction was diluted appropriately, and formalin was added to it with O,'
Added to a concentration of 1 to 1.2 V/v%, preferably 0.4 to 0.8 v/v, 20 to 43"C1, preferably 37 to 4
Process for 5-60 days at 0°C. By this formalin detoxification treatment, LPF activity and H8F activity (His
tamine-sensitizing facto
r) etc. will be reduced. The present inventors have found that if amino acids are added in this detoxification treatment, the time required for detoxification is significantly shortened, and no aggregate precipitation occurs and toxicity reversion phenomenon (reverse) does not occur. At this time, Tween 80 is used as a stabilizer along with the amino acids.
, gelatin can be added as appropriate. In the case of glutaraldehyde, it is desirable to add it to a concentration of 0.05-03% v/v and treat it at room temperature for 1-7 days.

アミノ酸としては、グリシン、メチオニン、シスナイン
、グルタミン酸ナトリウム、アスパラギン酸、セリン、
アラニン、ロイシン、イソロイシン、バリン、スレオニ
ン、γ−アミノ酪酸１．リシンなどから１種または２種
以上が選ばれて用いられる。Amino acids include glycine, methionine, cisnine, monosodium glutamate, aspartic acid, serine,
Alanine, leucine, isoleucine, valine, threonine, γ-aminobutyric acid 1. One or more types are selected and used from ricin and the like.

シクロデキストリンあるいはその誘導体を添加した液状
培地で発酵槽を用いて得られる培養液から精製したＨＡ
画分を、上記のアミノ酸を添加してホルマリンで無毒化
する場合には、凝集塊沈殿を生成しないのでメンブラン
フィルタ−による無菌濾過を行なうことが可能である。HA purified from a culture solution obtained using a fermenter in a liquid medium supplemented with cyclodextrin or its derivatives
When the above-mentioned amino acids are added to the fraction and the fraction is detoxified with formalin, sterile filtration using a membrane filter can be performed since no aggregate precipitate is produced.

一方、アミノ酸を実質的に存在しない状態でホルマリン
無毒化処理を適用すると、シクロデキストリンを添加し
ない液状培地で静置培養して得られる培養液から精製し
たＨＡ画分を無毒化する場合と同様に、凝集塊沈殿を生
じ、以後の工程において音波処理によってこの沈殿を破
砕する必要がある。この場合無毒化後の工程における無
菌濾過は困難である。On the other hand, when formalin detoxification treatment is applied in the substantial absence of amino acids, the same results as when detoxifying the HA fraction purified from the culture solution obtained by static culture in a liquid medium without cyclodextrin. , resulting in agglomerate precipitation, which needs to be broken up by sonication in subsequent steps. In this case, sterile filtration in the step after detoxification is difficult.

上記無毒化処理後、適当な蛋白濃度に調製（通常、最終
蛋白窒素濃度８〜２０μｐ’Ｉ’ｃＡＰＮ揖）し、所望
によりさらにジフテリアトキソイド、破傷風トキソイド
を加え、そのまままたは必要にょシアシュバントとして
水酸化アルミニウムまたはリン酸アルミニウムを最終濃
度０１５〜０．３μｇ４４程度に加えて処理する。最後
に安定剤としてゼラチン、グルコース、保存剤としてチ
メロサールなどを適当量加えてワクチンとする。After the above detoxification treatment, the protein concentration is adjusted to an appropriate level (usually, the final protein nitrogen concentration is 8 to 20 μp'I'cAPN), and if desired, diphtheria toxoid and tetanus toxoid are further added, and aluminum hydroxide is added as is or as a dioxidase band if necessary. Alternatively, treatment is performed by adding aluminum phosphate to a final concentration of about 0.15 to 0.3 μg44. Finally, appropriate amounts of gelatin and glucose as stabilizers and thimerosal as a preservative are added to prepare the vaccine.

実施例 つぎに実験例および実施例を挙げて本発明をさらに具体
的に説明するが本発明はこれらに限定されない。EXAMPLES Next, the present invention will be explained in more detail with reference to experimental examples and examples, but the present invention is not limited thereto.

実施例 ５０／？の発酵槽（丸菱理化株製）に、下記第１表に示
す組成を有する改良培地にメチル化β−ＣＤを最終濃度
１．０９／ｌになるように添加した培地３５１を加え、
百日ぜき菌Ｉ相菌を１．　□　ＩＯＵ／ｍ７の量で接種
し、スパージャ−にょる槽底がらの通気攪拌培養系でＤ
Ｏの制御範囲を種々変え、温度３５℃、ｐＨ７，２に制
御し、消泡手段として機械的消泡を用い、それぞれ２４
時間培養を行なった。Example 50/? To a fermenter (manufactured by Marubishi Rika Co., Ltd.), add medium 351, which is an improved medium having the composition shown in Table 1 below, with methylated β-CD added to a final concentration of 1.09/l,
Pertussis I phase bacteria 1. □ D.
The control range of O was varied, the temperature was controlled to 35°C, the pH was controlled to 7.2, and mechanical defoaming was used as the defoaming means.
Time culture was performed.

第１表 →基礎培地は１２１℃、３０分間高圧滅菌し、補液は濾
過淋菌し、使用前に両者を混合して用いる。Table 1 → The basal medium is autoclaved at 121° C. for 30 minutes, and the replacement solution is filtered with Neisseria gonorrhoeae and mixed before use.

得られた培養液について、先と同様にして菌数を測定し
、またＦ−ＨＡを血球凝集試験（５ａｔｏ。Regarding the obtained culture solution, the number of bacteria was measured in the same manner as above, and the F-HA was tested using a hemagglutination test (5ato.

Ｙ、　ｅｔ、　ａｌ、　Ｉｎｆｅｃｔ、　Ｉｍｍｕｎ、
　７．９２９〜９９９．１９７３を参照）により測定、
ＬＰＦ−ＨＡをｉｎ　ｖｉｔｒｏ　ｆは■ｐ−ＥＬＩＳ
Ａ法（佐原ら、第２８回毒素シンポジウム予稿集、１４
１〜１４４．１９８１を参照）による単位（Ｌ　Ｐ　Ｅ
　ｕ／ｍｌと略記する）を測定し、ｉｎ　ｖｉｖｏでは
ｄｄ／Ｙマウス（４週令、雌）を用いＬＰＦ−ＨＡ静静
注３後後白血球数をカウントする方法（銘木ら、第２９
回毒素シンポジウム予稿集、１〜５．１９８２を参照）
によって測定した。その結果を第２図に示す。Y, et, al, Infect, Immun,
7.929 to 999.1973),
LPF-HA in vitro f p-ELIS
Method A (Sahara et al., Proceedings of the 28th Toxin Symposium, 14
1-144.1981) units (L P E
(abbreviated as u/ml), and in vivo, using dd/Y mice (4 weeks old, female) to count the number of white blood cells after 3 intravenous injections of LPF-HA (Meiki et al., No. 29).
)
Measured by. The results are shown in FIG.

第２図から明らかなように、菌増殖ならびにＨＡ画分の
高単位の産生が見られるのはＤｏが０７〜６０ｐｐｍで
あり、ＤＯ１，０〜５．５　ｐｐｍではとくに良好な結
果が得られた。As is clear from Figure 2, bacterial growth and high-unit production of HA fraction were observed at Do of 07 to 60 ppm, and particularly good results were obtained at DO of 1.0 to 5.5 ppm. .

比較実験例１〜１０および実験例２〜５培養条件を種々
かえて百日ぜき菌ＨＡ画分の産生量を比較検討した。す
なわち、従来の通気攪拌を一定とする方法と本発明に基
づく通気攪拌を連続的に変化させる方法について比較し
た。Comparative Experimental Examples 1 to 10 and Experimental Examples 2 to 5 The production amount of the Bordetella pertussis HA fraction was comparatively studied by varying the culture conditions. That is, a comparison was made between a conventional method in which aeration and agitation are kept constant and a method in which aeration and agitation are continuously varied based on the present invention.

１、４１の通気攪拌培養装置（Ｎ’ＢＳ社製）に、実験
例１で用いたものと同じメチル化β−ＣＤを最終濃度１
．０ｆｉ／ＩＪ添加した改良培地１０１を加え、百日ぜ
き菌Ｉ相菌を１．ＱＩＯ則頷ｌの量で接種し、スパージ
ャ−による槽底からの通気攪拌培養系で、第２表に示す
条件下に、すべて３５℃で３６時間培養した。Methylated β-CD, the same as that used in Experimental Example 1, was added to a final concentration of 1 in an aerated agitation culture device (manufactured by N'BS) in No. 1 and 41.
．． Improved medium 101 supplemented with 0fi/IJ was added, and Bordetella pertussis I phase bacteria were added to 1. The cells were inoculated in an amount equivalent to the QIO rule, and cultured at 35° C. for 36 hours under the conditions shown in Table 2 in an aerated culture system with aeration from the bottom of the tank using a sparger.

通気攪拌を一定とし消泡処理を行なわない培養は第２表
中の比較実験例（以下単に比較例という）１〜５，７お
よび８である。その中では比較例５の１０　Ｏｒｐｍで
Ｑ、５ｒｒｍという条件がＨＡ画分の産生量は良好であ
った。しかしながら、その場合においても培養１０時間
まではある程度の対数増殖（約１０　ＩＯＵ／ｍ７　）
を示したが、その後急激に増殖速度が減じ３６時間後に
おいても約１５ＩＯＵΔ扉にとどまり、ＨＡＡ分産生量
はＦ　　ＨＡが１６Ｈ’　Ａ　／ｍｅ　、　Ｌ　Ｐ　Ｆ
　−ＨＡ　カ１００　ＩＯＵ／ｍｄ　テキわめて低く、
４８時間後１で培養を続けた場合においてもほとんど増
加しなかった。Comparative Experimental Examples (hereinafter simply referred to as Comparative Examples) 1 to 5, 7, and 8 in Table 2 are cultures in which aeration and agitation are constant and no defoaming treatment is performed. Among them, the conditions of Comparative Example 5 of 10 Orpm, Q, and 5 rrm produced a good amount of HA fraction. However, even in that case, there is a certain degree of logarithmic growth (approximately 10 IOU/m7) until 10 hours of culture.
However, the proliferation rate decreased rapidly and remained at about 15 IOUΔ even after 36 hours, and the HAA production amount was 16H'A/me for FHA, LPF
-HA 100 IOU/md Very low,
Even when the culture was continued after 48 hours, there was almost no increase.

また、０．２　ｖｖｍで通気し、攪拌を５００　ｒｐｍ
あるいは６００　ｒｐｍの一定とし、消泡処理をしない
培養系は、比較例７，８であるが、いずれの場合も培養
後５〜１０時間で、激しい発泡のために菌体が槽壁土部
に付着するが培養液が槽外へ流失し、それを３６時間後
にすべて回収混合した場合においてもＨＡ画分量はきわ
めて低かった。Also, aeration at 0.2 vvm and stirring at 500 rpm.
Alternatively, the culture systems with a constant speed of 600 rpm and no antifoam treatment are Comparative Examples 7 and 8, but in both cases, bacterial cells adhered to the tank wall soil due to intense foaming 5 to 10 hours after culture. However, even when the culture solution flowed out of the tank and was collected and mixed after 36 hours, the amount of HA fraction was extremely low.

ＤＯを制御するが消泡処理を行なわない培養系の比較例
１０においても、同様の培養液の流失や菌体の槽壁への
付着がおこシ、ＨＡ画分量は低かった。In Comparative Example 10, a culture system in which DO was controlled but no defoaming treatment was performed, similar flow-off of the culture solution and adhesion of bacterial cells to the tank wall occurred, and the amount of HA fraction was low.

通気攪拌を一定とし、がっ消泡処理を行なう培養系は比
較例６および９である。いずれの場合も、培養初期のＤ
ＯがＨＡＡ分産生にとって不適当な６．０ｐｐｍ以上に
あり、培養の進行に伴ないＤＯは連続的に下降しつづけ
３６時間以前に菌増殖およびＨＡ産生に不適当な０．７
　ｐｐｍ以下の状態に達していた。本発明の方法を一部
加味して化学的消泡処理を行なった比較例９では、３６
時間培養後にオイテ８０　ＩＯＵ／ｍＡに達したが、Ｆ
−ＨＡは１２８ＨＡ／ｆｎｌ、ＬＰＦ−、ＨＡｉｊ５Ｑ
ＱＬＰＥＵ／−程度であった。Comparative Examples 6 and 9 are culture systems in which aeration and agitation are constant and defoaming treatment is performed. In either case, D
O is at 6.0 ppm or more, which is inappropriate for HAA production, and DO continues to decrease as the culture progresses, and before 36 hours, it reaches 0.7 ppm, which is inappropriate for bacterial growth and HA production.
It had reached a state of less than ppm. In Comparative Example 9, in which chemical defoaming treatment was carried out in part by the method of the present invention, 36
After incubation for an hour, it reached 80 IOU/mA, but F
-HA is 128HA/fnl, LPF-, HAij5Q
It was about QLPEU/-.

一方、培養液中のＤＯをＤｏコントローラー（ＮＢＳ社
製）を用いて自動的に連続的に通気量あるいは攪拌速度
を変化させて、１６〜３．５　ｐｐｍとなるように制御
しかつ消泡処理をしながら培養を行なったものが実験例
２〜５であるが、それらは培養１０時間後においても対
数増殖を維持し、最終的には３６時間できわめて高い菌
数、Ｆ−ＨＡ量およびＬ　Ｐ　Ｆ　−ＨＡ量を得ること
ができた。On the other hand, DO in the culture solution was controlled to 16 to 3.5 ppm by automatically and continuously changing the aeration amount or stirring speed using a Do controller (manufactured by NBS), and antifoaming treatment was performed. In Experimental Examples 2 to 5, the culture was carried out under the following conditions, and they maintained logarithmic growth even after 10 hours of culture, and finally reached extremely high bacterial counts, F-HA amounts, and L at 36 hours. The amount of PF-HA could be obtained.

なお、上方からの表面通気による方法では、ＨＡ画分の
産生は全く認められなかった。In addition, in the method using surface ventilation from above, no production of HA fraction was observed.

これらの成績から明らかなように、発酵槽を用いた通気
攪拌培養では、ＤＯ非制御下においては消泡処理をした
場合に菌増殖は認められるが、それらの例ではＦ−ＨＡ
およびＬＰＦ−４Ａはいずれも産生量が低いことがわか
った。一方、ＤＯ制御下で行った場合には菌増殖のみな
らずＦ−ＨＡ量およびＬＦＦ−ＨＡ量ともに著しく増大
した。As is clear from these results, in aerated agitation culture using a fermenter, bacterial growth is observed when antifoaming is applied under non-DO control, but in these examples, F-HA
It was found that the production amounts of both LPF-4A and LPF-4A were low. On the other hand, when the test was carried out under DO control, not only bacterial growth but also the amount of F-HA and LFF-HA significantly increased.

このように発酵槽を用いた通気攪拌培養では、ＤＯ制御
によって菌増殖は勿論、目的とする百日ぜき菌ＨＡ産生
量の著しい増大が図れることが判明した。As described above, it has been found that in the aerated agitation culture using a fermenter, not only bacterial growth but also a significant increase in the desired amount of Bordetella pertussis HA production can be achieved by controlling DO.

比較実験例１１ならびに実験例６および７上述のように
、本発明による特定の条件制御下に培養することによシ
目的とする百日ぜき菌ＨＡ両分の大量産生が達成される
が、これを従来公知の静置培養における場合と比較する
と第３表に示すとおシである。なお、表に示す各培養の
条件は下記のとおりである。ただし、°菌接種量と培養
温度はそれぞれ１．　Ｑ　ＩＯＵ／ｍｄ　、および３５
°ｃで共通とした。Comparative Experimental Example 11 and Experimental Examples 6 and 7 As described above, the desired mass production of both Bordetella pertussis HA can be achieved by culturing under specific controlled conditions according to the present invention. Table 3 shows the results compared to known static culture. The conditions for each culture shown in the table are as follows. However, the amount of bacteria inoculated and the culture temperature are 1. Q IOU/md, and 35
The temperature was set as common at °C.

（Ａ）静置培養（比較例１１） 培養容器ニル−瓶、１．５１容 培地：実施例１で用いたものと同じ改良培地ｏ２培養時
間＝１２０時間 （至）制御培養（本発明の方法）（実験例６および７） 培養容器：３００ｎ容発酵槽（丸菱理化製）培地：実験
例１で用いたものと同じ改良培地、２００１　；メチル
化β〜ＣＤ（実験例６）またはメチル化α−ＣＤ（実験
例７）１、Ｏｇ／４を添加 ＤＯ副制御　２．２−２．４　ｐｐｍ 消泡：機械的消泡手段（回転ディスク方式による） ｐＨ制御：　ｐＨ７，３ 培養時間：３５時間 上記結果を第３表に示す。(A) Static culture (Comparative Example 11) Culture container Ni-bottle, 1.51 volume Medium: Same improved medium as used in Example 1 o2 Culture time = 120 hours (up to) Controlled culture (method of the present invention) ) (Experimental Examples 6 and 7) Culture container: 300 n capacity fermenter (manufactured by Marubishi Rika) Medium: The same improved medium used in Experimental Example 1, 2001; Methylated β to CD (Experimental Example 6) or Methylated Addition of α-CD (Experiment Example 7) 1, Og/4 DO sub-control 2.2-2.4 ppm Defoaming: Mechanical defoaming means (by rotating disk method) pH control: pH 7.3 Culture time: 35 Time The above results are shown in Table 3.

第３表 第３表の結果からも明らかなように、本発明方法によれ
ば従来静置法に比べて菌増殖およびＬＰＦ−ＨＡ量とも
に著しく増大しておシ、Ｆ−ＨＡ量は同等かそれ以上で
、例えば菌数は２〜３倍、ＬＰＦ−ＨＡ量は１０倍以上
増大し、培養時間は１２０時間から３５時間へと大幅に
短縮されている。As is clear from the results in Table 3, the method of the present invention significantly increases both the bacterial growth and the amount of LPF-HA compared to the conventional static method. For example, the number of bacteria increases by 2 to 3 times, the amount of LPF-HA increases by more than 10 times, and the culture time is significantly shortened from 120 hours to 35 hours.

なお、実験例６および７におけるＤＯ制御下での培養の
場合の菌数、Ｆ−ＨＡ量およびＬＰＦ−ＨＡ量の経時的
な推移を第３図（Ｉ）および（Ｉｌ）、第４図（Ｉ）お
よび■にそれぞれ示した。これらの図からも明らかなよ
うに、本発明によるＤＯ制御下に培養した場合には菌数
の増大とともにＦ−ＨＡ量およびＬＰＦ−４Ａ量も著し
く増大される。In addition, the time course of the number of bacteria, amount of F-HA, and amount of LPF-HA in the case of culture under DO control in Experimental Examples 6 and 7 is shown in Figures 3 (I) and (Il) and Figure 4 ( I) and ■ are shown respectively. As is clear from these figures, when cultured under DO control according to the present invention, as the number of bacteria increases, the amount of F-HA and LPF-4A also increases significantly.

実施例１ 前記実験例１で得られたＨＡ画分を蛋白濃度が３０μｆ
　Ｔ　ＣＡ　Ｐ　Ｎ／ｍｌとなるように希釈し、これに
ホルマリン０．６または１．　Ｏｖ／Ｖ％の濃度に添加
し、３７℃または３９℃で５〜２１日間処理する。この
ホルマリンによる無毒化処理の際、第４表に示す各種ア
ミノ酸を表示の濃度にて添加する。Example 1 The HA fraction obtained in Experimental Example 1 was adjusted to have a protein concentration of 30 μf.
Dilute to T CA P N/ml and add formalin 0.6 or 1. Add to a concentration of Ov/V% and treat at 37°C or 39°C for 5-21 days. During this detoxification treatment with formalin, various amino acids shown in Table 4 are added at the indicated concentrations.

この無毒化処理の際の凝集塊沈殿の有無、また無毒化処
理後、透析チューブを用いて透析してホルマリンを除去
したのち、マウス毒性を検査しもさらに、３７℃で３週
間加温して毒性復帰現象の有無を検査した。また得られ
たワクチンの力価も測定した。それらの結果を第４表に
示す。The presence or absence of aggregate precipitation during this detoxification treatment was examined, and after the detoxification treatment, the formalin was removed by dialysis using a dialysis tube, and mouse toxicity was examined. The presence or absence of toxicity reversion was examined. The titer of the obtained vaccine was also measured. The results are shown in Table 4.

２１ 実施例２ 百日ぜき菌Ｉ相菌東浜株をポルデーシャ２グ培地で継代
したものを種菌とし、これをメチル化β−Ｃ’Ｄ　１０
　ｒｒＬ９／ｍｌを添加した実験例１で用いたものと同
じ改良培地０．４１を入れた２１容三角コルベンに、菌
数の最終濃度１．　□　ＩＯＵ、４Ａで接種し、これを
３５℃で１８時間培養して元培養菌を得だ。21 Example 2 The inoculum was prepared by subculturing Bordetella pertussis I phase fungus Higashihama strain in Pordesha 2g medium, and the methylated β-C'D 10
In a 21-volume triangular Kolben containing 0.41 of the same improved medium used in Experimental Example 1 with the addition of 9/ml of rrL, the final concentration of bacteria was 1. □ Inoculated with IOU, 4A, and cultured at 35°C for 18 hours to obtain original culture.

上記と同じ培地２００１を入れた３００１容槽型発酵槽
（丸菱理化製）に上記元培養菌を１，０■（１）べ接種
し、通気攪拌培養する。A 3001 fermenter (manufactured by Marubishi Rika) containing the same medium 2001 as above was inoculated with 1.0 μg (1) of the above-mentioned original culture, and cultured with aeration and stirring.

この培養はＤＯＩ、８〜２．７ｐｐｍになるように通気
量と攪拌速度の双方を自動制御した。まだ培養温度３５
℃、　ｐｉｆ　７．２に自動制御し、さらに機械的消泡
（回転ディスク方式）を行なった。In this culture, both the aeration amount and the stirring speed were automatically controlled so that the DOI was 8 to 2.7 ppm. Still culture temperature 35
℃ and pif 7.2, and mechanical defoaming (rotating disk method) was performed.

３５時間培養後、培養液を採取する。このようにして得
られた培養液について菌数およびＨＡ画分量を測定した
ところ、菌数２１０　ＩＯＵ／ｍ７　、　Ｆ−ＨＡＡ量
１　Ｑ　２４　ＨＡ　／ｍｌ　、　Ｌ　Ｐ　Ｆ　−ＨＡ
量２，４００ＬＰＥＵ／ｍｌであった。After culturing for 35 hours, the culture solution is collected. When the number of bacteria and the amount of HA fraction were measured for the culture fluid thus obtained, the number of bacteria was 210 IOU/m7, the amount of F-HAA was 1 Q24 HA/ml, and the amount of LPF-HA
The amount was 2,400 LPEU/ml.

上記で得られた培養液を遠心して上清を集め、その上清
に硫酸アンモニウムを謁飽和になるように加え、生じた
沈殿を１０．ＯＯＯｒｐｍ　３０分間遠心して集め、こ
れを１モル塩化ナトリウム添加リン酸緩衝液（、ｐｉｆ
　７．２　）に溶解し、不溶の沈殿部分を１０．００　
Ｏｒｐｍ、３０分間遠心して除去した。この上清に硫酸
アンモニウムを約１カ飽和に加え、生じた沈殿を同様に
遠心分離して集め、再び上記と同じ緩衝液に溶解、透析
チューブに入れて４℃で透析し不溶の沈殿部分を同様に
遠心分離して除去した。これを超遠心にかけ、得られだ
上清をさらに１０〜３０％シヨ糖密度勾配遠心（３９，
０００ｒｐｍｓ２０時間）にかけて、その上清（ＨＡ画
分）を回収した。これをメンブランフィルタ−にて除菌
した。このＨＡ画分の蛋白濃度を３０μｇＴＣＡ　Ｐ　
Ｎ／ｍｌに上記と同じ緩衝液を用いて調製した。The culture solution obtained above was centrifuged to collect the supernatant, ammonium sulfate was added to the supernatant to saturation, and the resulting precipitate was collected in 10. OOOrpm was collected by centrifugation for 30 minutes, and then diluted with phosphate buffer supplemented with 1M sodium chloride (pif
7.2), and the undissolved precipitate was dissolved in 10.00
Orpm and centrifuged for 30 minutes to remove. About 1 ton of ammonium sulfate was added to this supernatant to saturation, and the resulting precipitate was similarly centrifuged and collected, dissolved again in the same buffer as above, placed in a dialysis tube, dialyzed at 4°C, and the undissolved precipitate was removed in the same manner. was removed by centrifugation. This was subjected to ultracentrifugation, and the resulting supernatant was further centrifuged with 10-30% sucrose density gradient (39,
000 rpms for 20 hours), and the supernatant (HA fraction) was collected. This was sterilized using a membrane filter. The protein concentration of this HA fraction was adjusted to 30 μg TCAP.
N/ml was prepared using the same buffer as above.

なお、上記採取した培養液の処理工程およびＨＡ画分の
精製工程はおもに２〜４°Ｃにて行なった。Incidentally, the treatment process of the collected culture solution and the purification process of the HA fraction were mainly performed at 2 to 4°C.

得られ九Ｈ，Ａ画分にホルマリンを０．６　ｖ／ｖ％、
ツイーン８０を０．０５　ｖ／ｖ％、ゼラチンを０．０
２Ｗ／Ｖ％およびグリシンを０．２５　Ｍ加え、３９°
Ｃで７日間加温した。0.6 v/v% formalin was added to the obtained nine H and A fractions,
Tween 80 at 0.05 v/v%, gelatin at 0.0
Add 2W/V% and 0.25 M glycine, 39°
It was heated at C for 7 days.

０、７　ｗ／ｖ％塩化ナトリウム添加リン酸す衝ｆｉ（
ｐＨ’７．２　）に対して透析チューブに入れて透析し
てホルマリンを除去し、これを上記緩衝液にて蛋白濃度
８μｆＴＣＡＰＮ／ｍｇと彦るように希釈し、無毒化Ｈ
Ａ画分希釈液を得だ。0,7 w/v% sodium chloride added phosphoric acid (
The formalin was removed by dialysis against pH'7.2) in a dialysis tube, and this was diluted with the above buffer solution to a protein concentration of 8 μf TCAPN/mg.
A diluted fraction A solution was obtained.

この希釈液に水酸化アルミニウムゲルを０．２０ｍｆ／
ｍｌ（アルミニウム換算）になるように加えてＨＡ画分
を吸着させた。これにさらに保存剤としてチメロサール
０．０１　ｗ／ｖ％添加して沈降精製百日ぜきワクチン
を調製した。Add aluminum hydroxide gel to this diluted solution at 0.20mf/
ml (in terms of aluminum) to adsorb the HA fraction. Further, 0.01 w/v% of thimerosal was added as a preservative to prepare a precipitated and purified pertussis vaccine.

このワクチンは国家検定規準（生物学的製剤基準、薬発
第２８７号、１９８１を参照）に従って検定を行なった
ところ、第５表に示すとおりすべて適合していることが
判明した。This vaccine was tested in accordance with the national testing standards (see Biological Product Standards, Yakuhatsu No. 287, 1981) and was found to comply with all the requirements as shown in Table 5.

第５表 実施例３ 前記実施例１で得られた無毒化ＨＡ画分希釈液に、ジフ
テリアトキソイド３３Ｌｆ／ｍおよび破傷風トキソイド
５Ｌｆ７ｍを添加し、これに水酸化アルミニウムゲルを
０．２０　ｔｎｙ／ｍｌ　（アルミニウム換算）加えて
ゲル吸着させた。これに安定剤としてゼラチン０．０２
　ｗ／ｖ　％　、ブドウ糖０．１　ｗ／ｖ　％、保存剤
としてチメロサール０．０１　ｗ／ｖ％を加え、沈降精
製百日ぜき・ジフテリア・破傷風混合ワクチンを調製し
た。Table 5 Example 3 Diphtheria toxoid 33Lf/m and tetanus toxoid 5Lf7m were added to the detoxified HA fraction diluted solution obtained in Example 1, and aluminum hydroxide gel was added at 0.20 tny/ml ( (in terms of aluminum) and gel adsorption. Add 0.02 gelatin as a stabilizer.
% w/v, glucose 0.1 w/v %, and thimerosal 0.01 w/v % as a preservative were added to prepare a precipitated and purified pertussis-diphtheria-tetanus combination vaccine.

この混合ワクチンは国家検定規準（生物学的製剤基準、
薬発第２８７号、１９８１を参照）に従って検定を行な
ったところ、第６表に示すとお９すべて適合しているこ
とが判明した。This combination vaccine meets national certification standards (biological product standards,
When the test was carried out in accordance with Yakuhatsu No. 287, 1981), it was found that all nine items shown in Table 6 were in compliance.

【図面の簡単な説明】[Brief explanation of drawings]

第１図は百日ぜき菌の増殖と培養温度の関係を示すグラ
フ、第２図は培養液中のＤＯ制御範囲と２４時間培養後
の菌数、Ｆ−ＨＡ量およびＬＰＦ−ＨＡ量を示すグラフ
、第３図（Ｉ）および（ＩＩ）ならびに第４図（Ｉ）お
よび（ＩＩ）はＤＯ２，２〜２．４１）ｐｍの制御下に
培養した場合の菌数、Ｆ−ＨＡ量およびＬＰＦ−ＨＡ量
の経時的な推移を示すグラフである。 −２８： 手続補正書（睦） 昭和５８年６、ａ１３日 特許庁長官　殿 １事件の表示 昭和５８年特許願第　０５８５４８　　５２、発明の名
称 百日ぜきワクチンの製造方法 ３、補正をする者 事件との関係　特許出願人 住所　熊本県熊本市清水町大窪６６８番地名称　財団法
人化学及血清療法研究所 （ほか１名） ４代理人 ５補正命令の日付　自発 （Ｉ）明則書第１〜２頁の特許請求の範囲を別紙のとお
り補正する。 ■同書の「発明の詳細な説明」のＳ″ｆｆ：下記のとお
り補正する。 （１）２頁末行：　「ＬＰＥ−Ｉ−ＩＡｊを「ＬＰＩｉ
’−ＨＡ」と補正。 （２〕５頁下から３行：　「ＨＡ画分」をｊＦ−ＨＡ画
分」と補正。 （３）８頁末行：「通常の不活化剤、例えば」を削除。 （４〕８頁末行〜９頁１行：「やグルタルアルデヒド」
を削除。 （５）９頁下から３〜２行二「やグルタルアルデヒドな
ど」を削除。 （６）１６頁７行：「５〜６０日間」全「３〜６０日間
ｊと補正。 ｔ７Ｈ６頁下から５〜３行二Ｆグルタルアルデヒドの・
・・（中略）・・・が望ましい。」を削除。 （８０９頁第１表中ｌＯ行ニドリスヒドロキンメチルア
ミノメタンの含量のｒｌ、５２５」”ｋ　ｒ６１００Ｊ
と補正。 （９］２２頁７行：　「培養後５〜１０時間」を「培養
５〜１０時間」と補正。 ＱＱ３０頁第４頁中４表中段：「アルギニン」の項を全
行削除。 ｑυ３１頁第４表（続き）中：「グリンン＋アルギニン
」の項を全行削除。 以上 補正した特許請求の範囲 （１）百日ぜき菌をシクロデキストリンまたはその誘導
体を添加した液状培地に接種し、培養温度２０〜３７℃
で培地の溶存酸素量を０７〜６．０　ｐｐｍの範囲に保
ちかつ消泡処理をしながら通気攪拌培養し、対数増殖期
ないし定常期の菌発育段階で感染防御抗原）ＩＡ画分を
採取し、ついで該ＨＡ画分をアミノ酸の存在下に無毒化
することを特徴とする百日ぜきワクチンの製造方法。 （２＞　−ｙ　ミノ酸がグリシン、メチオニン、システ
ィン、グルタミン酸ナトリウム、アスパラギン酸、セリ
ン、７ラニン、ロイシン、インロイシン、バリン、スレ
オニン、γ−アミノ酪酸、リジンから選ばれる１＠また
は２種以上でおる前記第（１〕項の方法。 （３）液状培地がカザミノ酸を０．１〜２０Ｐ／ｌ、ア
スコルビンｅ’ｋｏ、０１〜１９／ｌ、グルタチオンを
Ｏ１〜５（１／／およびシフロブキス１−リンまたはそ
の誘導体を０．００１〜５グ／ｌ含有している前記第（
１１項の方法。 （４）シクロデキストリンまたはその誘導体がメチル化
α−シクロデキストリン、メチル化β−ンクロテキスト
リン、メチル化γ−シクロデキストリン、α−ンクロデ
キストリン、β−ンクロデキストリンおよびγ−シクロ
デキストリンから選ばれる１種または２種以上である前
記第（１）項の方法。 （５）培養時間を７〜４０時間とする前記第（１）項の
方法。 （６］消泡処理が、機械的消泡手段、化学的消泡剤の添
加またはそれらの組合わせによる前記第（１）項の方法
。 （７）ｐＨが６０〜９０の範囲である前記第（１）項の
方法。Figure 1 is a graph showing the relationship between the growth of Bordetella pertussis and culture temperature, Figure 2 is a graph showing the DO control range in the culture solution, the number of bacteria after 24-hour culture, the amount of F-HA, and the amount of LPF-HA. Figure 3 (I) and (II) and Figure 4 (I) and (II) show the number of bacteria, amount of F-HA, and LPF-HA when cultured under the control of DO2, 2 to 2.41) pm. It is a graph showing the change in amount over time. -28: Procedural Amendment (Mutsu) June 13, 1980 Director General of the Patent Office Representation of Case 1 Patent Application No. 058548 52, Title of Invention Process for Producing Pertussis Vaccine 3, Person Making Amendment Related Patent Applicant Address 668 Okubo, Shimizu-cho, Kumamoto City, Kumamoto Prefecture Name Chemo and Serum Therapy Research Institute (and 1 other person) Date of 4th agent 5th amendment order Voluntary (I) Patent claim on pages 1-2 of the Memorandum of Regulations Correct the range as shown in the attached sheet. ■S″ff of “Detailed Description of the Invention” in the same book: Amended as follows. (1) Bottom line of page 2: “LPE-I-IAj has been changed to “LPIi
Corrected to '-HA'. (2] 3 lines from the bottom of page 5: Corrected “HA fraction” to “jF-HA fraction”. (3) Last line of page 8: Delete “common inactivating agent, e.g.”. (4) Page 8 End line - page 9, line 1: "Ya glutaraldehyde"
Delete. (5) Delete 3-2 lines 2 "and glutaraldehyde, etc." from the bottom of page 9. (6) Page 16, line 7: "5 to 60 days" All corrected to "3 to 60 days j."
... (omitted) ... is desirable. ” was deleted. (Rl of the content of nidrishydroquine methylaminomethane in row 10 of Table 1 on page 809, 525"k r6100J
and correction. (9) Page 22, line 7: Corrected “5 to 10 hours after culturing” to “5 to 10 hours of culture.” QQ Page 30, page 4, table 4 middle row: Delete the entire line “Arginine”. qυ Page 31 In Table 4 (Continued): All rows of "Grin + Arginine" are deleted. Claims as amended above (1) Bordetella pertussis is inoculated into a liquid medium supplemented with cyclodextrin or its derivatives, and cultured at a temperature of 20 - 37℃
While maintaining the amount of dissolved oxygen in the medium in the range of 0.7 to 6.0 ppm and performing antifoaming treatment, the culture was carried out with aeration and agitation, and the IA fraction (protective antigen) was collected during the logarithmic growth phase or stationary phase of bacterial growth. and then detoxifying the HA fraction in the presence of amino acids. (2> -y The amino acid is one or more selected from glycine, methionine, cysteine, monosodium glutamate, aspartic acid, serine, 7-lanine, leucine, inleucine, valine, threonine, γ-aminobutyric acid, and lysine) The method according to item (1) above. (3) The liquid medium contains 0.1 to 20 P/l of casamino acids, 01 to 19 P/l of ascorbine e'ko, 01 to 5 P/l of glutathione (1// and 1 - containing 0.001 to 5 g/l of phosphorus or its derivatives (
Method of Section 11. (4) The cyclodextrin or its derivative is one selected from methylated α-cyclodextrin, methylated β-ncrotextrin, methylated γ-cyclodextrin, α-ncrodextrin, β-ncrodextrin, and γ-cyclodextrin. Or the method of item (1) above, which is two or more types. (5) The method of item (1) above, wherein the culture time is 7 to 40 hours. (6) The method according to item (1) above, wherein the defoaming treatment is performed by adding a mechanical defoaming means, a chemical defoaming agent, or a combination thereof. The method described in (1).

Claims (7)

【特許請求の範囲】[Claims] （１）百日ぜき菌をシクロデキストリンまたはその誘導
体を添加しだ液状培地に接種し、培養温度２０〜３７℃
で培地の溶存酸素量を０．７〜６．０　ｐｐｍの範囲に
保ちかつ消泡処理をしながら通気攪拌培養し、対数増殖
期々いし定常期の菌発育段階で感染防御抗原ＨＡ画分を
採取し、ついで該ＨＡ画分をアミノ酸の存在下に無毒化
することを特徴とする百日ぜきワクチンの製造方法。(1) Bordetella pertussis was inoculated into a liquid medium supplemented with cyclodextrin or its derivatives, and the culture temperature was 20-37°C.
The amount of dissolved oxygen in the culture medium was kept in the range of 0.7 to 6.0 ppm, and the culture was carried out with aeration and agitation while antifoaming treatment was performed, and the infectious protective antigen HA fraction was extracted during the logarithmic growth phase to the stationary bacterial growth stage. A method for producing a pertussis vaccine, which comprises collecting the HA fraction, and then detoxifying the HA fraction in the presence of an amino acid. （２）アミノ酸がグリシン、メチオニン、システィン、
グルタミン酸ナトリウム、アスパラギン酸、セリン、ア
ラニン、ロイシン、インロイシン、バリン、スレオニン
、γ−アミノ酪酸、リジンから選ばれる１種または２種
以上である前記第（１）項の方法。(2) Amino acids are glycine, methionine, cysteine,
The method according to item (1) above, wherein one or more selected from monosodium glutamate, aspartic acid, serine, alanine, leucine, inleucine, valine, threonine, γ-aminobutyric acid, and lysine. （３）液状培地がカザミノ酸を０，１〜２０ｆ／１１，
１スコルビン酸を００１〜１１／ｌ、グルタチオンを０
．１〜５　Ｑ　ｆ／１２およびシクロデキストリンまた
はその誘導体をｏ、ｏｏｉ〜５　ｆ／／ｌ含有している
前記第（１）項の方法。(3) The liquid medium contains casamino acids of 0.1 to 20 f/11,
1 Scorbic acid 001-11/l, glutathione 0
．． 1 to 5 Q f/12 and cyclodextrin or a derivative thereof in an amount of o, ooi to 5 f//l. （４）シクロデキストリンまたはその誘導体がメチル化
α−シクロデキストリン、メチル化α−シクロデキスト
リン、メチル化γ−シクロデキストリン、α−シクロデ
キストリン、β−シクロデキストリンおよびｒ−シクロ
デキストリンから選ばれる１種または２種以上である前
記第（１）項の方法。(4) The cyclodextrin or its derivative is one selected from methylated α-cyclodextrin, methylated α-cyclodextrin, methylated γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin, and r-cyclodextrin, or The method according to item (1) above, which is two or more types. （５）培養時間を７〜４０時間とする前記第（１）項の
方法。(5) The method of item (1) above, wherein the culture time is 7 to 40 hours. （６）消泡処理が、機械的消泡手段、化学的消泡剤の添
加またはそれらの組合わせによる前記第（１）項の方法
。(6) The method according to item (1) above, wherein the defoaming treatment is performed by adding a mechanical defoaming means, a chemical defoaming agent, or a combination thereof. （７）　ｐｌ＋が６．０〜９．０の範囲である前記第（
１）項の方法・(7) The above-mentioned (
1) Method・